Field of the Invention
The present invention relates to methods, devices, and systems for calibrating a temperature of one or more microfluidic channels in a microfluidic device. More particularly, aspects of the present invention relate to methods, devices, and systems for using an amplicon having a known or expected thermal melting temperature to calibrate a thermal sensor and/or thermal control element used to control the temperature of a microfluidic channel in a microfluidic device. The present invention also relates to methods, devices and systems for calibrating the temperature of a thermal melt in a microfluidic channel of a microfluidic device. More particularly, aspects of the present invention relate to methods, devices, and systems for using a calibrant present in a test sample and amplifying the calibrant to produce a calibrant amplicon in which the calibrant amplicon has a known or expected thermal melting temperature to calibrate the melting temperature of an amplicon of a nucleic acid of interest. The present invention also relates to the use of a calibrant present in a test sample as a control for determining whether amplification of a sample has occurred.
Description of the Background
Devices for performing chemical, biological, or other reactions (e.g., a microfluidic device for performing polymerase chain reaction (PCR) amplification of DNA molecules, or a microfluidic molecular diagnostic platform that performs PCR on a patient sample and then uses the PCR product for genotyping by performing a high resolution melt analysis) often feature one or more thermal control elements that are used to subject reactants to a desired thermal profile. A description of PCR amplification, and an example of one possible microfluidic device including thermal control elements for PCR amplification and thermal melt analysis, are provided in U.S. Patent Application Publication Nos. 2009/0248349 and 2011/0056926, the entire disclosures of which are incorporated herein by reference.
In many applications of such microfluidic devices (e.g., PCR and/or thermal melt analysis), the thermal control elements of those devices must be precisely calibrated. That is, the correspondence between the temperature of the thermal control element and an electrical characteristic of the thermal control element must be precisely determined. For example, in the case of a resistance temperature detector and/or heater, the correspondence between temperature and resistance must be precisely determined. Additional types of thermal control elements can include platinum resistive heaters, thermistors, diode temperature sensors, thermocouples, or any other suitable temperature measuring devices. Additional electrical characteristics of thermal control elements that correspond to temperature can include capacitance or inductance of an element, frequency, pulse width, or amplitude of a signal, or other sensor characteristics known in the art.
Among many variations that can occur in microfluidic devices, temperature variation between channels is particularly relevant. Securing the right and uniform temperature among channels in a microfluidic device or chip leads to a reliable and consistent PCR and thermal melt analysis. In turn, having a successful amplification and melt of the target sequence may lead to a correct sample genotyping, which may lead to the characterization of a patient with a genetic disorder or a genetic predisposition or the efficiency of drug metabolism.
As noted above, assuring that microfluidic channels in a chip present the same temperature during a PCR and thermal melt analysis reaction will result in improved reliability and consistency. However, with such small elements and such small volumes of reagents, it is very challenging to test and prove that channels at the same time are performing a PCR plus thermal melting under the same optimal temperature conditions.
Methods of calibrating thermal control elements of a microfluidic device often include generating a lookup table or a series of coefficients that define a calibration equation, i.e., a lookup table or an equation relating the temperature of the thermal control element with the electrical characteristic.
Calibration can be performed by sending the device to a third party laboratory for taking accurate measurements and generating the lookup table or series of coefficients; however, this procedure is generally expensive and time consuming. Furthermore, for many devices (e.g., many common microfluidic devices) there may be many thermal control elements (e.g. dozens or even hundreds of heaters and sensors), each of which requires its own precise calibration, making third-party calibration impractical.
Accordingly, there is a need in the art for a reliable calibrant that will be useful in calibrating the temperature of one or more microfluidic channels in a microfluidic device or chip. Similarly, there is a need for robust calibration of thermal sensors and/or heaters that can be accurate, reduce downtime and maintain high throughput.